Assay for Detection of Transient Intracellular CA2+

ABSTRACT

This invention relates to a simple end point assay for detection of transient intracellular Ca 2+  with broad applicability to many Ca 2+  channel proteins comprising, Generation of expression constructs for the fusion proteins having the Ca 2+ /calmodulin dependent protein kinase II (CaMKII) phosphorylation sites of NR2A or NR2B subunits of N-methyl-D-aspartate receptor (NMDAR) or the voltage gated potassium channel of  Drosophila  (Eag) or any protein sequence which binds to the T-site of CaMKII similar to NR2B, conjugated to mitochondrial localizing signal sequence, or mutants of these sequences as described herein. Generation of mammalian expression constructs of α-CaMK11 as a chimera with green fluorescent protein (GFP-α-CaMKII) or its mutants as described herein. Site-Directed mutagenesis, Transfection, Ca 2+  stimulation, imaging and quantification of the number of cells with Ca 2+ -dependent signal, wherein, NMDA receptor activity assay, TRPVI receptor activity assay, GluR4 receptor activity assay are performed to detect the activity of Ca 2+  channel proteins.

This application is a divisional application of co-pending U.S. patentapplication Ser. No. 13/661,787, filed Oct. 26, 2012, which is acontinuation-in-part application of U.S. patent application Ser. No.12/665,599, filed Jun. 9, 2010, now U.S. Pat. No. 8,304,198, which isthe U.S. national phase of PCT Application No. PCT/IN08/00370, filed onJun. 12, 2008, which claims priority to Indian Patent Application No.1276/CHE/07, filed on Jun. 19, 2007, all of which are incorporated byreference herein in their entirety.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 159826 ST25.txt. The size of the textfile is 1,241 bytes, and the text file was created on Oct. 14, 2015

FIELD OF INVENTION

The invention relates to detection and quantification of transientchanges in intracellular metabolites by making use of a resultingprotein-protein interaction that is irreversible and longlasting.

BACKGROUND OF INVENTION

Increase in concentration of intracellular free Ca²⁺ ions is a widelyused signaling mechanism under various physiological conditions. Releaseof free Ca²⁺ ions inside the cell happens either through plasma membranechannel proteins that allow Ca²⁺ influx or by release from intracellularCa²⁺ stores. Since Ca²⁺ release thus becomes the activity of a widevariety of proteins, methods for detection and measurement ofintracellular free Ca²⁺ are used as assays for many proteins that arepotential drug targets. These methods should be capable of sensing thetransient rise in intracellular [Ca²⁺] and also of generating adetectable signal.

Most of the commonly used methods for Ca²⁺ sensing use molecules thatexhibit changes in their fluorescence characteristics upon exposure toincrease in [Ca²⁺]. These include small molecular weight macromoleculessuch as fura-2, indo-1, fluo-3, and Calcium-Green (U.S. Pat. Nos.4,603,209 and 5,049,673) as well as proteins such as GFP (greenfluorescent protein) family of proteins with a sensor peptide insertedwithin them (U.S. Pat. No. 6,469,154) or the cameleon molecules oftandem GFP constructs with an intervening Ca²⁺ sensing peptide sequence,that generate a FRET signal upon sensing free Ca²⁺ (U.S. Pat. No.5,998,204). In all these methods, the fluorescence signal is also asshort-lived as the Ca²⁺-transient, and hence signal acquisition inreal-time is mandatory. Moreover this also imposes rapid liquiddispensing. These factors not only necessitates the use of moreexpensive equipments but also limits the choice of equipments that canbe used in addition to imposing severe constraints for carrying outthese assays at high throughput scale. U.S. Pat. No. 6,514,709 discloseda method that prolongs the duration of the Ca²⁺-transients byincorporating an intracellular chelator that alters the kinetics ofsignal generation, so that the signal is prolonged. However in all thesemethods, measurements will have to be done on cells in the livecondition.

OBJECTS OF THE INVENTION

The main object of this invention is to detect intracellular Ca²⁺transients using simple end point assay.

The other object is to develop a Ca²⁺ sensing assay with broadapplicability to many Ca²⁺ channel proteins.

Another object is to detect intracellular Ca²⁺ transients using a simplemethod wherein the need for realtime measurements on live cells isavoided.

STATEMENT OF INVENTION

This invention relates to a simple end point assay for detection oftransient intracellular Ca²⁺ with broad applicability to many Ca²⁺channel proteins comprising, Generation of expression constructs for thefusion proteins having the Ca²⁺/calmodulin dependent protein kinase II(CaMKII) phosphorylation sites of NR2A or NR2B subunits ofN-methyl-D-aspartate receptor (NMDAR) or the voltage gated potassiumchannel of Drosophila (Eag) or any protein sequence which binds to theT-site of CaMKII similar to NR2B, conjugated to mitochondrial localizingsignal sequence, or mutants of these sequences as described herein.Generation of mammalian expression constructs of a- CaMKII as a chimerawith green fluorescent protein (GFP-α-CaMKII) or its mutants asdescribed herein. Site-Directed mutagenesis, Transfection, Ca²⁺stimulation, imaging and quantification of the number of cells withCa²⁺-dependent signal, wherein, NMDA receptor activity assay, TRPVIreceptor activity assay, GluR4 receptor activity assay are performed todetect the activity of Ca²⁺ channel proteins.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a schematic representation of the assay system for detectionof release of intracellular free Ca²⁺;

FIG. 2 shows Ca²⁺-dependent generation of signal due to GFP-α-CaMKIItranslocation towards mitochondrially localized NR2B sequence (MLS-NR2B)observed as the appearance of punctae;

FIG. 3 shows Ca²⁺-dependent generation of signal due to GFP-α-CaMKIItranslocation towards full-length NR2B localized on intracellularmembranous organelles;

FIG. 4 shows two images of cells, one obtained after inducingCa²⁺-influx to cells (Image 1) and the other obtained without inducingany Ca²⁺-influx (Image 2);

FIG. 5 shows the qunatitation data obtained as described in FIG. 4, forthe wild-type and T286D mutant forms of GFP-α-CaMKII;

FIG. 6 shows the quantitation data obtained as described in FIG. 4, forthe wild-type and T286D mutant forms of GFP-α-CaMKII, at differentconcentrations of Ca²⁺ in the extracellular medium;

FIG. 7 shows Ca²⁺-dependent generation of signal due to GFP-α-CaMKIItranslocation towards mitochondrially localized Eag sequence (MLS-Eag);

FIG. 8 shows signal generated due to Ca²⁺-influx through NMDA receptorformed of NR1 and NR2B subunits;

FIG. 9 shows signal generated due to Ca²⁺-influx through NMDA receptorformed of NR1 and NR2A subunits;

FIG. 10 shows signal generated due to Ca²⁺-influx through vanilloidReceptor (TRPV1)

FIG. 11 shows signal generated due to Ca²⁺-influx through the AMPA-typeglutamate receptor, G1uR4;

FIGS. 12A-12B show (A) Cavaα1c+GFP CaMKII transfected cells activatedwith ionomycin and calcium and (B) Cavaα1c+GFP CaMKII transfected cellswithout ionomycin in the presence of calcium;

FIG. 13 shows HEK-293 cells co-transfected with NR1 and NR2C subunits ofNMDA receptor along with GFP-α-CaMKII and MLS-NR2B, unless indicatedotherwise, (A) without activation, (B) activated with glutamate in thepresence of glycine and calcium, (C) activated with NMDA in the presenceof glycine and calcium, (D) activated with glutamate in the presence ofglycine and EGTA, (E) activated with glutamate in the presence ofglycine, calcium, and MK801, and (F) cells without NR1 and NR2C subunitstransfected therein activated with glutamate in the presence of glycineand calcium;

FIG. 14 shows HEK-293 cells co-transfected with NR1 and NR2D subunits ofNMDA receptor along with GFP-α-CaMKII and MLS-NR2B, unless indicatedotherwise, (A) without activation, (B) activated with glutamate in thepresence of glycine and calcium, (C) activated with NMDA in the presenceof glycine and calcium, (D) activated with glutamate in the presence ofcalcium, (E) activated with glutamate in the presence of glycine,calcium, and MK801, and (F) cells without NR1 subunit transfectedtherein activated with glutamate in the presence of glycine and calcium;

FIG. 15 shows HEK-293 cells co-transfected with G1uR1, GFP-α-CaMKII andMLS-NR2B activated by (A) glutamate or (C) AMPA in the presence ofcalcium and activated by (B) glutamate or (D) AMPA in the presence ofcalcium and CNQX;

FIG. 16 shows HEK-293 cells co-transfected with G1uR2, GFP-α-CaMKII andMLS-NR2B activated by (A) glutamate or (C) AMPA in the presence ofcalcium and activated by (B) glutamate or (D) AMPA in the presence ofcalcium and CNQX;

FIG. 17 shows HEK-293 cells co-transfected with G1uR3,GFP-T286D-α-CaMKII and MLS-NR2B activated by (A) glutamate or (C) AMPAin the presence of calcium and activated by (B) glutamate or (D) AMPA inthe presence of calcium and CNQX;

FIG. 18 shows HEK-293 cells transfected with GFP-α-CaMKII (A-F),MLS-NR2B (A, C-F) and the cDNA for P2X2 channel (B-F) activated in thepresence of (A, B, F) calcium and ATP, (C) ATP alone or, (D) calciumalone. FIG. 18(E) shows cells fixed at time=0;

FIGS. 19A-19B show (A) activation of HEK-293 cells transfected with VGCCsubunits (Cavaα1c and β subunits) together with GFP-α-CaMKII andMLS-NR2B by BayK8644 and, (B) HEK-293 cells transfected with VGCCsubunits (Cavaα1c and β subunits) together with GFP-α-CaMKII andMLS-NR2B without activation by BayK8644;

FIG. 20 shows construction of the pcDNA 3.1 Zeo vector with MLS-NR2B andGFP-α-CaMKII;

FIGS. 21A-21C show HEK-293 cells transfected with the vector shown inFIG. 20 under zeocin selection shown through (A) bright fieldmicroscopy, (B) detection GFP fluorescence, and (C) an overlay of brightfield microscopy and GFP fluorescence; and

FIG. 22 shows specificity of the calcium signal seen when cellstransfected with both MLS-NR2B and GFP-α-CaMKII through the vector shownin FIG. 20 are activated with ionomycin and calcium.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an assay for detecting intracellularbinding between two proteins. The assay is useful for detectingintracellular release of free Ca²⁺. In this assay method the transientCa²⁺ signal is captured by an irreversible protein- protein interactionso that the signal is converted into a form that can be preserved fordays to even months. There is absolutely no requirement of any real timesignal acquisition or live cell signal acquisition.

Increase in concentration of intracellular free Ca²⁺ ions is a widelyused signaling mechanism under various physiological conditions. Releaseof free Ca²⁺ ions inside the cell happens either through plasma membranechannel proteins that allow Ca²⁺ influx or by release from intracellularCa²⁺ stores. Since Ca²⁺ release thus becomes the activity of a widevariety of proteins, methods for detection and measurement ofintracellular free Ca²⁺ are used as assays for many proteins that arepotential drug targets. These methods should be capable of sensing thetransient rise in intracellular [Ca²⁺] and also of generating adetectable signal.

Most of the commonly used methods for Ca²⁺ sensing use molecules thatexhibit changes in their fluorescence characteristics upon exposure toincrease in [Ca²⁺]. These include small molecular weight macromoleculessuch as fura-2, indo-1, fluo-3, and Calcium-Green (U.S. Pat. Nos.4,603,209 and 5,049,673) as well as proteins such as GFP (greenfluorescent protein) family of proteins with a sensor peptide insertedwithin them (U.S. Pat. No. 6,469,154) or the cameleon molecules oftandem GFP constructs with an intervening Ca²⁺ sensing peptide sequence,that generate a FRET signal upon sensing free Ca²⁺ (U.S. Pat. No.5,998,204). In all these existing methods, the fluorescence signal isalso as short-lived as the Ca²⁺-transient, and hence signal acquisitionin real-time is mandatory. This requires sophisticated equipments withincreased temporal resolution and capability for real time measurementsfrom live cells. Moreover this also imposes rapid liquid dispensing.Consequently the cost of instrumentation becomes significantly high aswell as the choice of equipments that can be used becomes limited. Inaddition severe constraints are imposed for carrying out these assays athigh throughput scale. U.S. Pat. No. 6,514,709 disclosed a method thatprolongs the duration of the Ca²⁺-transients by incorporating anintracellular chelator that alters the kinetics of signal generation, sothat the signal is prolonged. Even then, measurements will have to bedone on cells in the live condition.

Ca²⁺/calmodulin dependent protein kinase type II (CaMKII) present atneuronal synapses, upon activation by increase in intracellular Ca²⁺,gets translocated to the NMDA-type glutamate receptor and bindsirreversibly by its T-site (the Thr²⁸⁶-binding site) to the NR2B subunitof the receptor. A GFP-fusion of CaMKII (GFP-CaMKII) and a fusion of thebinding sequence on NR2B with a mitochondrial targeting sequence(MLS-NR2B) upon co-transfection in HEK-293 cells, bind to each other inresponse to increase in intracellular Ca²⁺. GFP-CaMKII present in thecytosol translocates to mitochondria, where it binds to MLS-NR2B. FIG. 1depicts schematically the appearance of punctae of green fluorescence incells which had a uniform distribution of green fluorescence beforestimulation with Ca²⁺. To demonstrate this phenomenon, intracellularCa²⁺ influx was generated by adding the ionophore, ionomycin, whichbinds to the cell membrane and forms pores through which extracellularCa²⁺ flows in. This is a combination of two published methods (Strack,S., McNeill, R. B., and Colbran, R. J., 2000, J Biol Chem 275,23798-23806; Bayer, K. U., LeBel, E., McDonald, G. L., O'Leary, H.,Schulman, H. and De Koninck, P., 2006, J. Neurosci. Vol. 26, p1164-1174)but is not identical to either.

Surprisingly it has been found that the percentage of green cells thatformed green punctae increases with increase in concentration ofextracellular Ca²⁺ provided. At higher concentrations of extracellularCa²⁺, more cells formed green punctae. Since extracellular concentrationof Ca²⁺ is related to the concentration of intracellular Ca²⁺ levels inthe system, it is possible to quantitate intracellular free Ca²⁺concentration.

Since the signal from fluorescent Ca²⁺-sensing dyes is also as shortlived as the increased Ca²⁺ levels, the commonly used methods requiresophisticated equipments with increased temporal resolution andcapability for real time measurements from live cells. This not onlynecessitates the use of more expensive equipments but also limits thechoice of equipments that can be used. For example in case offluorescence microscope, only inverted microscope could be used. On thecontrary, the interaction between CaMKII and NR2B is irreversible andhence produces a stable signal corresponding to a transient increase inintracellular Ca²⁺ thereby eliminating the necessity for real timemeasurements. After completing the experiment on cover slips in multiwell cell culture plates, the cells can be fixed and mounted on slidesand can be viewed in an upright or inverted fluorescence microscope.Since the method disclosed in the present invention is not dependent onreal time data acquisition techniques it is useful in drug discoveryprogram by adapting it for high throughput screening for compounds thatinhibit proteins which cause increase in intracellular [Ca²⁺] such asCa²⁺-channel proteins. Moreover, the method disclosed in the presentinvention could enable many more laboratories with lesserinstrumentation, to take up investigations on events and proteins thatcause increase in intracellular [Ca²⁺].

GFP-α-CaMKII was constructed in the vector, pEGFP of Clontech, using thecDNA of CaMKII obtained as a gift from Prof. Mary B. Kennedy of theDivision of Biology, California Institute of Technology, Pasadena, USA(Praseeda M., Pradeep K. K., Krupa A., Krishna S. S., Leena S., Kumar R.R., Cheriyan J., Mayadevi M., Srinivasan N. and Omkumar R. V., 2004,Biochem J, 378, 391-397).

MLS-NR2B was constructed in the vector pSGH, obtained as gift from Dr.Stefan Strack, Department of Pharmacology, University of Iowa, USA(Strack, S., McNeill, R. B., and Colbran, R. J. (2000) J Biol Chem 275,23798-23806) using the cDNA of NR2B obtained as a gift from Prof. S.Nakanishi, Graduate School of Medicine and Faculty of Medicine, KyotoUniversity, Department of Biological Sciences, Kyoto, JAPAN. The cDNAencoding NR2A also was a gift from Prof. S. Nakanishi, Department ofBiological Sciences, Kyoto University, JAPAN. The mammalian expressionvectors for NR1 (pRc-NR1), NR2A and NR2B (pRK-NR2A and pRK-NR2B) weregifts from Dr. David Lynch, Children's Hospital of Philadelphia, USA.The cDNA of vanilloid receptor type 1 (VR1), also known as TRPV1(Caterina et al., 1997, Nature, Vol. 389, p816-824) was a provided byDr. David Julius, Department of Physiology, University of California SanFransisco, San Fransisco, Calif. 94158-2517, USA. The cDNA of glutamatereceptor, was provided by Prof. Peter Seeberg, Department of MolecularNeurobiology, Max Planck Institute for Medical Research, D-69120Heidelberg, Germany.

Experimental Procedures

Generation of Expression Constructs for the Fusion Proteins having thePhosphorylation Sites on NR2A or NR2B, Conjugated to MitochondrialLocalizing Signal Sequence

The cDNAs of NR2A (encoding amino acid residues 1265-1301) or NR2B(encoding amino acid residues 1271-1311) harboring the phosphorylationsite for CaMKII (Ser¹²⁹¹ and Ser¹³⁰³ of NR2A and NR2B respectively)present between the BamHI and EcoRI sites in pGEX-2T-NR2A orpGEX-2T-NR2B vectors (Praseeda, M., Mayadevi, M., and Omkumar, R. V.,2004, Biochem. Biophys. Res. Commun. 313, 845-849) were subcloned intothe pSGH vector. The sequences of NR2A and NR2B form parts of fusionproteins with a mitochondrial targeting signal sequence, a myc-epitopeand GST at the N-terminal end (MLS-NR2A or MLS-NR2B). The sequences ofthe fusion constructs were confirmed by DNA sequencing of the NR2A orNR2B segments.

Generation of Mammalian Expression Constructs of α-CaMKII

The cDNA of α-CaMKII was amplified by PCR from pGEM2α (Praseeda M.,Pradeep K. K., Krupa A., Krishna S. S., Leena S., Kumar R. R., CheriyanJ., Mayadevi M., Srinivasan N. and Omkumar R. V., 2004, Biochem J, 378,391-397) using forward primer having nucleotide sequence as set forth inSEQ ID NO: 1 and reverse primer having nucleotide sequence as set forthe in SEQ ID NO: 2 to yield a PCR product with KpnI and BamHI sites at5′ and 3′ ends respectively. The PCR product was cloned into pEGFP-C1vector (Clontech) using the corresponding restriction sites in themultiple cloning site of the vector, which resulted in an N-terminalGFP-fusion of α-CaMKII (GFP-α-CaMKII).

5′-CGGGGTACCATGGCTACCATCACCTGC-3′ SEQ ID NO: 15′-CGCGGATCCCTATCAATGGGGCAGGACGG-3′ SEQ ID NO: 2

Site-Directed Mutagenesis

Site-directed mutagenesis was carried out using the QuikChangeSite-Directed Mutagenesis Kit from Stratagene based on Kunkel's method(Kunkel, T. A., 1985, Proc. Natl. Acad. Sci. USA 82, 488-492). The cDNAof α-CaMKII present in the expression vector pEGFP-C1-α-CaMKII served asthe template to generate mutants. The NR2A or NR2B sequences present inthe pSGH-NR2A or pSGH-NR2B expression vectors were also used astemplates to generate mutants.

Transfection, Ca²⁺-Stimulation and Imaging

HEK-293 (Human Embryonic Kidney) cells were cultured as monolayer inDMEM containing 10% FBS and antibiotics (penicillin 100 units/ml,streptomycin 100 vg/ml, fungizone 2.5 μg/ml) at 37° C. in a humidatmosphere having 5% CO₂.

For transfection, 1.5×10⁴ HEK-293 cells were seeded on sterile 12 mmcover slips in 24 well plates (Nunc). After 24 hrs, the cells werecotransfected with plasmid constructs encoding GFP-α-CaMKII (0.1μg/well) and either of MLS-NR2A or MLS-NR2B (0.25 μg/well), or pRK-NR2Aor pRK-NR2B (along with pRc-NR1 for expression of functional NMDAreceptor) using Lipofectamine reagent. About 48 hrs followingtransfection, cells were washed with Hank's balanced salt solution(HBSS) containing 20 mM HEPES and 1.4 mM EGTA. The cells were thenincubated with 15 μM ionomycin in the same buffer for 10 min.Subsequently the cells were incubated in the same buffer containing 2 mMCaCl₂ and 3 μM ionomycin. Cells were fixed with 3.7% paraformaldehyde inPBS (Phosphate-buffered saline, 10 mM disodium hydrogen phosphate, 1.8mM potassium dihydrogen orthophosphate, 140 mM sodium chloride, 2.7 mMpotassium chloride, pH adjusted to 7.4) at different time points afteraddition of calcium, mounted with mountant containing DABCO (antifade)and were observed through either a simple fluorescence microscope or aconfocal fluorescence microscope (TCS -SP2-AOBS from Leica).

Appearance of perinuclear granules due to mitochondrial localization ofGFP-α-CaMKII is taken as the signal for binding between GFP-α-CaMKII andMLS-NR2B. In case of binding between GFP-α-CaMKII and full-length NR2B,accumulation of GFP-fluorescence in the membraneous structures such asnuclear membrane, endoplasmic reticulum membrane, plasma membrane, etcis taken as signal for binding. Data presented are for cells fixedimmediately after incubation with 15 μM ionomycin (−Ca²⁺ influx) or 2-5minutes after Ca²⁺ stimulus (+Ca²⁺ influx). The total number of greenfluorescent cells and cells which develop the signal for binding werecounted manually for each microscopic field. Number of cells thatdevelop signal for binding is represented as percentage of total numberof green fluorescent cells (number of green fluorescent cells whichdevelop signal for binding + number of green fluorescent cells which donot develop signal) in a field. Average for at least three separatefields was obtained for each experiment. Mean±standard deviation of dataobtained from three such experiments is presented as values in inset inFIG. 2. Wherever images for lack of binding are presented, no cell wasfound to develop the perinuclear granules in repeated experiments andhence no quantitation data is presented.

NMDA Receptor Activity Assay

For expression of NMDA receptor (NMDAR), the cells were cotransfectedwith plasmid constructs encoding GFP-α-CaMKII (0.1 μg/well) and NR1(pRc-NR1 plasmid) and NR2B (pRK-NR2B plasmid) subunits of NMDAR (0.33μg/well of pRc-NR1 and pRK-NR2B) using Lipofectamine (Invitrogen). Whengrowth medium was added to the cells, it was supplemented with aninhibitor of NMDA receptor, either 1 mM of 2-amino-5-phosphovaleric acid(AP-5) or 10 μM MK-801. For detecting activity of NMDAR, about 48 hrsfollowing transfection, cells were washed twice with Ca²⁺ and Mg²⁺ freeHank's balanced salt solution (HBSS). The cells were then incubated withHBSS solution containing glutamate (10 μM) and glycine (2 μM), with orwithout CaCl₂ (2 mM) for about 5 minutes. Cells were fixed with 3.7%paraformaldehyde in PBS for about 10 to 15 minutes and were washedthrice with PBS. The fixed cells were mounted onto glass slides.

In experiments with NMDAR containing NR2A subunit, cells wereco-transfected with plasmid constructs encoding GFP-α-CaMKII (0.1μg/well) and NR1 (pRc-NR1 plasmid) and NR2A (pRK-NR2A plasmid) subunitsof NMDAR (0.33 μg/well of pRc-NR1 and pRK-NR2A) along with MLS-NR2B(0.33 μg/well of pSGH-NR2B) and growth medium was not supplemented withAP-5. NMDAR activity was detected as described in the previous section.

TRPV1 Receptor Activity Assay

For the expression of TRPV1, HEK-293 cells were cotransfected with TRPV1(pCDNA3-TRPV1, about 0.2 μg/well), MLS-NR2B (pSGH-MLS-NR2B, about 0.33μg/well) and GFP-CaMKII (pEGFP-CaMKII, about 0.1 μg/well) usinglipofectamine reagent. After 48 hours, the transfected cells weresubjected to TRPV1 activity assay. The growth medium was removed and thecells were washed with Ca²⁺ and Mg free HBSS containing 400 μM EGTA. Thereceptor was activated with 3 μM or 10 nM of capsaicin with 2 mM Ca²⁺ inHBSS without Mg²⁺ for 5 minutes. Control without Ca²⁺ during activationwas also performed. The inhibitor of TRPV1 receptor, capsazepine (50μM), when used, was preincubated for 3 minutes with cells in Ca²⁺ andMg²⁺-free HBSS followed by activation using the components as describedabove with 50 μM capsazepine. Cells were then fixed and were mountedonto glass slides.

GluR4 Receptor Activity Assay

HEK-293 cells were cotransfected with GluR4 (pRK5-GluR4, about 0.33μg/well), MLS-NR2B (pSGH-MLS-NR2B, about 0.33 μg/well) and GFP-CaMKII(pEGFP-CaMKII, about 0.1 μg/well) using lipofectamine reagent. Duringgrowth, the medium was supplemented with 50 μM CNQX (inhibitor of AMPAreceptor). After 48 hours, the transfected cells were subjected to GluR4activity assay. The growth medium was removed and the cells were washedtwice with Ca²⁺ and Mg free HBSS containing 2 mM HEPES and 400 μM EGTA.The GluR4 receptor was activated with HBSS solution containing 10 μMglutamate with or without 2 mM Ca²⁺ for about 5 minutes. The inhibitor,CNQX (50 μM), when used, was preincubated for 3 minutes with cells inCa²⁺ and Mg²⁺-free HBSS followed by activation using the components asdescribed above with 50 μM CNQX. Cells were then fixed and were mountedonto glass slides.

Basic System for Detection of Intracellular Ca²⁺-Release

The system described in FIG. 1 was imaged by confocal and non-confocalfluorescence microscopes and the images are presented in FIG. 2. Thearrows in the Plus-Ca²⁺ images indicate the cells which develop punctateappearance due to mitochondrial localization of GFP-α-CaMKII, consequentto intracellular rise in [Ca²⁺]. Quantification of cell count carriedout as described in FIG. 4 is shown in the confocal Plus-Ca²⁺ image.

Development of Ca²⁺-Dependent Signals by Using Full-Length NR2B Insteadof MLS-NR2B

The data in FIG. 3 is obtained by transfecting cells with full-lengthNR2B instead of MLS-NR2B. Since full length NR2B is localized to nuclearand other intracellular membranes, GFP-CaMKII localizes to thosemembraneous structures instead of mitochondria, upon sensing increase inintracellular [Ca²⁺].

Quantitation of the Intracellular Ca²⁺-Dependent Signal

The inventors have found that the number of cells that develop theGFP-clustering signal is dependent on the concentration of extracellularCa²⁺ and hence the intracellular Ca²⁺ (FIG. 6). It also depends on theefficiency of the binding between CaMKII and NR2B (FIG. 5). Theinventors developed a method to express quantitatively the strength ofthe signal as mentioned in Methods section (Methodology forTransfection, Ca²⁺-stimulation and imaging). Data in FIG. 4 is used todemonstrate this method.

In image 1 of FIG. 4, there are totally 11 cells expressing GFP of whichtwo cells (numbered 3 and 7) show clustering/aggregation of GFP as seenby regions of intense fluorescence. Percentage of cells showingclustering is obtained as 2*100/11=27%.

In image 2, no cells are found to have the GFP clustering, out of the 6cells expressing GFP.

WT-CaMKII vs T286D-CaMKII—Cell Count Comparison

The cell count determined as mentioned in methods section as well as inthe previous section, for the wild-type and T286D mutant forms ofGFP-α-CaMKII are presented in FIG. 5. The T286D mutation which is knownto enhance the Ca²⁺/calmoduin affinity of α-CaMKII also improved itsefficiency of binding in the intracellular binding system as seen by theincreased (more than two fold) cell count (FIG. 5).

[Ca²⁺]-Dependence of Cell Count—WT and T286D-α-CaMKII

Different concentrations of extracellular Ca²⁺ are expected to produceproportional increases in intracellular [Ca²⁺] when ionomycin-mediatedCa²⁺-influx was generated. This was reflected in the cell count obtainedfor different extracellular [Ca²⁺] (FIG. 6). As [Ca²⁺] increased, thecell count also increased. Moreover the T286D mutant of GFP-α-CaMKIIwhich showed enhanced signal compared to GFP-(WT)-α-CaMKII (FIG. 5) alsoshowed improved sensitivity to [Ca²⁺] as seen by response at lower[Ca²⁺] upto 2 nM. This observation of dependence of the signal on [Ca²⁺]is the key advancement in our invention making this method suitable forsensing intracellular [Ca²⁺] in a semiquantitative manner.

Mutants of α-CaMKII and NR2B or NR2A that generate a localization signalin response to intracellular release of Ca²⁺

The following pairs of mutants of GFP-α-CaMKII and either of NR2A orNR2B sequences when expressed in HEK-293 cells exhibited. Ca²⁺-dependentbinding and generation of localization signals. Some of these pairsgenerated signals more than the wild-type pair of GFP-α-CaMKII andMLS-NR2B. These could be used either as detection systems with improvedsensitivity or under cellular environments where the wild-type pairencounters technical problems such as faulty expression.

-   -   1. T286D-α-CaMKII and MLS-WT-NR2B    -   2. F293E/N294D-α-CaMKII and MLS-WT-NR2B    -   3. MLS-(S1303A)-NR2B and WT-α-CaMKII    -   4. S1303A-NR2B and WT-α-CaMKII    -   5. K42R/T286D-α-CaMKII and MLS-WT-NR2B    -   6. MLS-Delta^(IN)NR2A (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of        NR2A) and WT-α-CaMKII    -   7. MLS-(S1291A)/Delta^(IN) (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of        NR2A) NR2A and WT-α-CaMKII    -   8. MLS-Delta^(IN)NR2A (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of        NR2A) and T286D-α-CaMKII    -   9. Delta^(IN)NR2A (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of NR2A)        and T286D-α-CaMKII    -   10. MLS-K1292A-NR2B and WT-α-CaMKII        Interaction of the Binding Site on Eag with GFP-CaMKII for        Detection of Intracellular Release of Ca²⁺

The voltage gated potassium channel of Drosophila (Eag) that islocalised in synapses as well as in axons also interacts at the T-siteof CaMKII (Sun, X. X., Hodge, J. J., Zhou, Y., Nguyen, M., and Griffith,L. C. (2004) J Biol Chem 279, 10206-10214). The Eag amino acid residues731-803 (or longer or shorter sequences) which comprise the CaMKIIbinding motif was cloned into the pSGH vector to obtain an MLS-Eagfusion that could be targeted to mitochondria. When wild-type orsuitable mutant forms of GFP-CaMKII coexpressed with wild-type orsuitable mutant forms of MLS-Eag were activated with increase inintracellular Ca²⁺, the GFP-CaMKII should translocate to mitochondriagiving rise to stable green fluorescent punctae as observed in case ofMLS-NR2B. FIG. 7 shows the Ca²⁺-depednent localization ofGFP-(WT)-CaMKII to MLS-Eag. This system could also be a Ca²⁺-sensorsimilar to the CaMKII-NR2B system.

Any peptide sequence which binds to the T-site of CaMKII similar to NR2Bcould be used as a binding partner for CaMKII in a Ca²⁺-sensing system.

NMDA Receptor Activity

The neuronal N-methyl-D-aspartate (NMDA)-type glutamate receptor is aglutamate activated Ca²⁺-channel that has NR1 and NR2B as subunits.

When activated with glutamate and glycine it caused influx of Ca²⁺ fromthe extracellular medium leading to translocation of GFP-α-CaMKII toNR2B that is localized in the nuclear membrane, endoplasmic reticulumand plasma membrane. This signal, shown by arrows in FIG. 8, was notformed in the absence of extracellular Ca²⁺. The signal generation wasblocked by the NMDA receptor specific inhibitors, AP-5 and MK-801,further establishing that the signal was specific for the activity ofNMDA receptor.

Activity of NMDA Receptor Containing NR2A Subunit

Functional NMDAR can be formed by NR1 and NR2A subunit also instead ofthe NR1/NR2B combination. However, since the NR2A subunit does not bindCaMKII, a translocation signal consequent to influx of Ca²⁺ will not beobtained. By additionally transfecting the mitochondrially localizedMLS-NR2B sequence, it is possible to detect the activity of theNR2A-containing NMDAR also in a Ca²⁺-dependent manner. The arrows inFIG. 9 show mitochondrially translocated GFP-α-CaMKII.

Detection and Measurement of the Activity of Other Ca²⁺-Channels

Many other proteins with intracellular Ca²⁺-releasing activity such asCa²⁺-channel proteins (nicotinic acetylcholine receptor channel (GrandoS A, Horton R M, Mauro T M, Kist D A, Lee T X, Dahl M V., J Invest.Dermatol., 1996, 107, p412-418), the vanilloid receptor 1 (TRPV1) (WitteD G, Cassar S C, Masters J N, Esbenshade T, and Hancock A A. 2002, JBiomol Screen. 7, p466-475), G-protein coupled receptors (Fujii, R.,Hosoya, M., Fukusumi, S., Kawamata, Y., Habata, Y., Hinuma, S., Onda,H., Nishimura, O., and Fujino, M. 2000, J. Biol. Chem., 275,p21068-21074), etc. could be expressed in a heterologous cell culturesystem to generate specific Ca²⁺-channel activity. Activity of all suchproteins could also be detected if they are co-expressed with a suitablepair of wild-type or mutant forms of GFP-CaMKII and NR2B, NR2A, Eag orany other protein which binds to the T-site of CaMKII, as described inearlier sections.

Vanilloid Rececptor (TRPV1)

Vanilloid receptor is a Ca²⁺-channel that is activated by capsaicin, oneof the active principle of hot chili peppers (Caterina et al., 1997,Nature, Vol. 389, p816-824). We have found that the activity ofvanilloid receptor (TRPV1) could be detected by our invention forCa²⁺-sensing. HEK-293 cells cotransfected with TRPV1, GFP-CaMKII andMLS-NR2B upon activation with capsaicin (3 μM or 10 nM), developed greenpunctae in a Ca²⁺-dependent manner as signal for the Ca²⁺-channelactivity of TRPV1 receptor. The signal generation was specific fortransfection by TRPV1, activation by capsaicin and also for the presenceof Ca²⁺ in the extracellular medium. Signal generation due to activationof TRPV1 could be inhibited by the TRPV 1-specific inhibitor capsazepine(FIG. 10). All these results show that the activity of TRPV1 receptor isbeing detected by our invention.

Neuronal AMPA-Type Glutamate Receptor GluR4

The homomeric AMPA-type glutamate receptor formed of the GluR4 subunitcauses Ca²⁺-influx upon activation by glutamate (Cushing, et al., 1999,J. Neurosci. Methods. Vol. 90, p33-36). Upon transfection of HEK-293cells with the G1uR4 subunit along with GFP-CaMKII and MLS-NR2B, it waspossible to detect the Ca²⁺-channel activity of the receptor. Thereceptor was activated by treatment with glutamate which caused theformation of green punctae due to translocation of GFP-CaMKII tomitochondrially localized MLS-NR2B. Generation of the signal could beblocked by the specific inhibitor of the receptor, CNQX (FIG. 11).

System Using an Expression Construct for the Voltage-Gated CalciumChannel Subunit Cavα1c

FIG. 12 shows HEK-293 cells transfected with Cavaα1c+GFP CaMKIIactivated with (A) ionomycin and calcium and (B) activated with calciumwithout ionomycin.

Detection of the Activity of NMDA Receptor Having NR1 and NR2C Subunits

With reference to FIG. 13, HEK-293 cells were co-transfected with NR1and NR2C subunits of NMDA receptor along with GFP-α-CaMKII and MLS-NR2B(A-E) or without NR1/NR2C (F). About 48 hrs after transfection, theHEK-293 cells were activated with either glutamate (B) or NMDA (C) inthe presence of glycine and Ca²⁺. In the same experiment, transfectedcells were activated with (D) glutamate in the presence of glycine andEGTA without calcium or (E) glutamate in the presence of glycine,calcium, and MK801. As a control, cells that were not transfected withNR1 and NR2C were activated with glutamate in the presence of glycineand calcium.

Detection of the Activity of NMDA Receptor Having NR1 and NR2D Subunits

With reference to FIG. 14, HEK-293 cells were co-transfected with NR1and NR2D subunits of NMDA receptor along with GFP-α-CaMKII and MLS-NR2B(A-E) or without NR1 (F). About 48 hrs after transfection, the HEK-293cells were activated with either glutamate (B) or NMDA (C) in thepresence of glycine and Ca²⁺. In the same experiment, transfected cellswere activated with (D) glutamate in the presence of glycine and withoutcalcium or (E) glutamate in the presence of glycine, calcium, and MK801.As a control, cells that were not transfected with NR1 were activatedwith glutamate in the presence of glycine and calcium.

Detection of the Activity of GluR1 Homomer of AMPA Receptor

With reference to FIG. 15, HEK-293 cells were co-transfected with GluR1,GFP-α-CaMKII and MLS-NR2B. About 48 hours after transfection, the cellswere activated with (A) glutamate or (C) AMPA in the presence ofcalcium. Another set of transfected cells was activated by (B) glutamateor (D) AMPA in the presence of calcium and the specific AMPA inhibitorCNQX.

Detection of the Activity of GluR2 Homomer of AMPA Receptor

With reference to FIG. 16, HEK-293 cells were co-transfected with GluR2,GFP-α-CaMKII and MLS-NR2B. About 48 hours after transfection, the cellswere activated with (A) glutamate or (C) AMPA in the presence ofcalcium. Another set of transfected cells was activated by (B) glutamateor (D) AMPA in the presence of calcium and the specific AMPA inhibitorCNQX.

Detection of the Activity of GluR3 Homomer of AMPA Receptor

With reference to FIG. 17, HEK-293 cells were co-transfected with GluR3,GFP-T286D-α-CaMKII and MLS-NR2B. About 48 hours after transfection, thecells were activated with (A) glutamate or (C) AMPA in the presence ofcalcium. Another set of transfected cells was activated by (B) glutamateor (D) AMPA in the presence of calcium and the specific AMPA inhibitorCNQX.

P2X2 Receptor Assay

With reference to FIG. 18, HEK-293 cells were seeded on coverslips in a24-well plate and transfected with GFP-α-CaMKII, MLS-NR2B and the cDNAfor the purigenic receptor P2X ligand-gated (P2X2) ion channel. The P2X2channel binds to adenosine triphosphate (ATP) (the ligand for thisligand-gated channel) and is thought to be responsible for mediatingsynaptic transmission between neurons and between neurons and smoothmuscle. The cells transfected with GFP-α-CaMKII, MLS-NR2B, and P2X2 werewashed 36 hours afterwards with HBSS containing 1 mM HEPES and 0.5 mMEGTA for five minutes followed by treatment with 100 μM ATP in HBSS(with 1 mM HEPES and 2 mM calcium chloride) for five minutes. Cells notreceiving ATP or calcium treatment (FIG. 18C, 18D), or those nottransfected with P2X2 or MLS-NR2B (FIG. 18B) were used as negativecontrols.

FIG. 18 shows ATP-dependent calcium signaling in (A) cells transfectedwith both GFP-α-CaMKII and MLS-NR2B, but not P2X2, activated by ATP inthe presence of calcium; (B) cells transfected with GFP-α-CaMKII andP2X2, but not MLS-NR2B, activated buy ATP in the presence of calcium;(C) cells transfected with GFP-α-CaMKII, MLS-NR2B and P2X2 activated byATP without calcium; (D) cells transfected with GFP-α-CaMKII, MLS-NR2Band P2X2 in the presence of calcium but not ATP; (E) cells transfectedwith GFP-α-CaMKII, MLS-NR2B and P2X2 and fixed prior to any activationor exposure to calcium; and (F) cells transfected with GFP-α-CaMKII,MLS-NR2B and P2X2 activated by ATP in the presence of calcium. Theresults show the localization of the signal as punctae in cellstransfected with -α-CaMKII, MLS-NR2B and P2X2 (FIG. 18F). The number ofcells with punctae is an indication of calcium influx.

Voltage-Gated Calcium Channel Activity Assay

With reference to FIG. 19, HEK-293 cells were transfected withvoltage-gated calcium channel (VGCC) subunits (Cavaα1c and β subunits)together with GFP-α-CaMKII and MLS-NR2B. Those cells were then activatedby BayK8644, a VGCC agonist, with results shown in FIG. 19A. FIG. 19Bshows the same transfected cells, without activation by BayK8644. Cellswere fixed for imaging following activation with BayK8644 and in thecontrol condition, and appearance of punctae, based on extracellularCa²⁺ and transfected VGCC, was assessed. Punctae formation may beinhibited by administration of nifedipine, a specific inhibitor of VGCC.

Formation of Stable Calcium Sensor Clones

With reference to FIG. 20, the pcDNA 3.1 Zeo vector with MLS-NR2B andGFP-α-CaMKII was constructed by cloning the cDNAs for both MLS-NR2B andGFP-α-CaMKII from the parent vectors viz. pSGH-MLS-NR2B and pEGFP-CaMKIIrespectively into the pIRES vector (Clontech—Cat No: 631605). ThesecDNAs were inserted into the two multiple cloning sites flanking theinternal ribosome entry site (IRES) sequence. pSGH-MLS-NR2B was digestedusing enzymes Nhe I and Eco RI and MLS-NR2B so released was cloned intothe multiple cloning site A of pIRES vector between the same sites.GFP-α-CaMKII was amplified by PCR using pEGFP-CaMKII as the template,forward primer with Xba I restriction site and reverse primer with NotIrestriction site, amplified product was digested with the respectiveenzymes and cloned to the multiple cloning site B of pIRES vector.

SEQ ID NO: 3  (5′-GCTCTAGAATGGTGAGCAAGGGC-3′)-forward primer for GFPSEQ ID NO: 4  (5′-ATAAGAATGCGGCCGCTCATCAATGGGGCAGGACGGAGG-3′)-reverse primer for CaMKIIThe whole reading frame incorporating the single mRNA coding forMLS-NR2B-IRES-GFP-α-CaMKII was then excised from thepIRES-MLS-NR2B-GFP-α-CaMKII using restriction enzymes Nhe I and Not I.This was then cloned to the multiple cloning site of pc DNA 3.1 (+)(Zeo)(Invitrogen—Cat No: V860-20) to make the pc DNA3.1(+) (Zeo)MLS-NR2B-IRES-GFP-α-CaMKII.

HEK-293 cells were then seeded at a density of 250,000 cells per well ina six well culture plate. 48 hours later, cells were transfected withthe pc DNA3.1(+) (Zeo) MLS-NR2B-IRES-GFP-α-CaMKII using lipofectamine.Twenty-four hours after transfection, cells were selected by treatmentwith 10% DMEM including zeocin antibiotic at 300 μg/mL. After 48 hours,all the GFP expressing cells were separated out using fluorescenceactivated cell sorter (FACS) and maintained in medium containing thesame concentration of the antibiotic. Similarly after a session of 4FACS, spanning the period of a month, an all GFP positive cellpopulation was obtained (MLS-2B-I-GC-Zeo-MP), (MP: denotes mixedpopulation, I: IRES, GC: GFP-CaMKII). FIG. 21 shows transfected HEK-293cells under zeocin selection shown through (A) bright field microscopy,(B) GFP fluorescence, and (C) an overlay of both imaging methods.

The mixed population of stable cells were trypsinised and seeded at verylow density in 6-well culture plates. 24 hours later, attached singlecells were marked under a light microscope. After they formed colonies,each colony of cells was aseptically transferred using colony pickingmethod, into new wells where they multiplied and formed a homogenouspopulation/clone of cells. They were designated as MLS-2B-I-GC-Zeo-CL-1(Clone 1).

The clones were further checked for the specificity of calcium signal(FIG. 22). They showed calcium dependent punctae formation uponionomycin mediated calcium influx treatment which was absent in cellswhere no calcium treatment was given.

What is claimed is:
 1. A stable calcium sensor cell line comprising: aconstitutively expressed protein carrying a sequence that can bind tothe T-site of α-Ca²⁺/calmodulin dependent protein kinase II (CaMKII) ora fusion protein comprising any sequence that can bind to the T-site ofCaMKII conjugated to a mitochondrial localizing sequence; and a chimericprotein, the chimeric protein comprising α-CaMKII or a mutant thereofand a fluorescent protein.
 2. The stable calcium sensor cell line asclaimed in claim 1, further comprising at least one expression constructcomprising a calcium channel protein.
 3. The stable calcium sensor cellline as claim in claim 1, wherein the cell line is produced by: i)generating a construct encoding a protein carrying any sequence that canbind to the T-site of CaMKII or a fusion protein comprising any sequencethat can bind to the T-site of CaMKII conjugated to a mitochondriallocalizing sequence; and a chimeric protein, the chimeric proteincomprising α-Ca²⁺/calmodulin dependent protein kinase (CaMKII) or one ofits mutants and a fluorescent protein; and an IRES sequence between theabove mentioned chimeric protein and protein or fusion protein havingany sequence that can bind to the T-site of CaMKII; ii) generating anexpression vector with a unique antibiotic selection marker for theconstruct mentioned in item (i) by subcloning the construct mentioned initem (i) having a protein carrying any sequence that can bind to theT-site of CaMKII or a fusion protein comprising any sequence that canbind to the T-site of CaMKII conjugated to a mitochondrial localizingsequence; and a chimeric protein, the chimeric protein comprisingα-Ca²⁺/calmodulin dependent protein kinase (CaMKII) or a mutant thereofand a fluorescent protein; and an IRES sequence between the abovementioned chimeric protein and protein or fusion protein having anysequence that can bind to the T-site of CaMKII; iii) transfecting cellswith the expression vector generated as mentioned in item (ii); iv)subjecting the cells to antibiotic selection pressure using theantibiotic referred in item (ii); v) sorting all the cells expressingthe fluorescent protein mentioned in item (i) by fluorescence activatedcell sorter (FACS) technology and subjecting the sorted cells again toantibiotic selection pressure as mentioned in item (iv); vi) repeatingcycles comprising the procedures mentioned in item (iv) followed by item(v) until almost all cells are positive for the fluorescent protein; andsubjecting the cells obtained in item (vi) that stably express thefluorescent protein to clonal selection by seeding at low density andseparating colonies formed from single cells by colony picking method.4. An assay for quantitation of intracellular calcium wherein for theactivity assay of any calcium channel protein, at least one expressionconstruct of the calcium channel protein is transfected to the calciumsensor cell line of claim 1.